Lamin A/C Is Dispensable to Mechanical Repression of Adipogenesis

Mesenchymal stem cells (MSCs) maintain the musculoskeletal system by differentiating into multiple lineages, including osteoblasts and adipocytes. Mechanical signals, including strain and low-intensity vibration (LIV), are important regulators of MSC differentiation via control exerted through the cell structure. Lamin A/C is a protein vital to the nuclear architecture that supports chromatin organization and differentiation and contributes to the mechanical integrity of the nucleus. We investigated whether lamin A/C and mechanoresponsiveness are functionally coupled during adipogenesis in MSCs. siRNA depletion of lamin A/C increased the nuclear area, height, and volume and decreased the circularity and stiffness. Lamin A/C depletion significantly decreased markers of adipogenesis (adiponectin, cellular lipid content) as did LIV treatment despite depletion of lamin A/C. Phosphorylation of focal adhesions in response to mechanical challenge was also preserved during loss of lamin A/C. RNA-seq showed no major adipogenic transcriptome changes resulting from LIV treatment, suggesting that LIV regulation of adipogenesis may not occur at the transcriptional level. We observed that during both lamin A/C depletion and LIV, interferon signaling was downregulated, suggesting potentially shared regulatory mechanism elements that could regulate protein translation. We conclude that the mechanoregulation of adipogenesis and the mechanical activation of focal adhesions function independently from those of lamin A/C.

3D Printable and Computational Models of the Bone Marrow Mechanical Environment

Aged individuals and astronauts experience bone loss despite rigorous physical activity. Bone mechanoresponse is in part regulated by mesenchymal stem cells (MSCs). We reported that daily low intensity vibration (LIV) restores MSC proliferation in senescence and simulated microgravity models, suggesting reduced mechanical signal delivery to MSCs likely contributes to declining bone mechanoresponse. To this end, we have developed a 3D bone marrow analog which controls trabecular geometry, marrow mechanics and external stimuli. Finite element (FE) models of hydrogels, representing bone marrow, were generated using instantaneous compression (1000% strain/s, 20% strain) and relaxation experiments (100s) of both gelatin and hyaluronin-based hydrogels. Experimental and in silico vibration experiments using molded-gelatin wells (widths= 4 , 5, 6 and 8 mm) were performed under 1g acceleration, 100 Hz for FE model calibration. For MSC experiments, 0.25cmgyroid-based trabeculae of bone volume fractions (BV/TV) corresponding to adult (25%) and aged (13%) mice were printed using polylactic acid. MSCs encapsulated (1x106 cells/mL) in migration-permissive hydrogelswithin [sic] printed trabeculae were exposed to LIV (1g, 100 Hz, 1 hour/day). After 14 days, type-I collagen, Ki-67, f-actin (n=3/grp) were quantified for extracellular matrix composition, proliferation, and morphology and grouped with respect to the maximum von Mises strain for 13.5% and 25% BV/TV scaffolds using the calibrated FE models.

Parameter-Dependency of Low-Intensity Vibration for Wound Healing in Diabetic Mice

Chronic wounds in diabetic patients represent an escalating health problem, leading to significant morbidity and mortality. Our group previously reported that whole body low-intensity vibration (LIV) can improve angiogenesis and wound healing in diabetic mice. The purpose of the current study was to determine whether effects of LIV on wound healing are frequency and/or amplitude dependent. Wound healing was assessed in diabetic (db/db) mice exposed to one of four LIV protocols with different combinations of two acceleration magnitudes (0.3 and 0.6 g) and two frequencies (45 and 90 Hz) or in non-vibration sham controls. The low acceleration, low frequency protocol (0.3 g and 45 Hz) was the only one that improved wound healing, increasing angiogenesis and granulation tissue formation, leading to accelerated re-epithelialization and wound closure. Other protocols had little to no impact on healing with some evidence that 0.6 g accelerations negatively affected wound closure. The 0.3 g, 45 Hz protocol also increased levels of insulin-like growth factor-1 and tended to increase levels of vascular endothelial growth factor in wounds, but had no effect on levels of basic fibroblast growth factor or platelet derived growth factor-bb, indicating that this LIV protocol induces specific growth factors during wound healing. Our findings demonstrate parameter-dependent effects of LIV for improving wound healing that can be exploited for future mechanistic and therapeutic studies.

Low-intensity vibration restores nuclear YAP levels and acute YAP nuclear shuttling in mesenchymal stem cells subjected to simulated microgravity

Reducing the musculoskeletal deterioration that astronauts experience in microgravity requires countermeasures that can improve the effectiveness of otherwise rigorous and time-expensive exercise regimens in space. The ability of low-intensity vibrations (LIV) to activate force-responsive signaling pathways in cells suggests LIV as a potential countermeasure to improve cell responsiveness to subsequent mechanical challenge. Mechanoresponse of mesenchymal stem cells (MSC), which maintain bone-making osteoblasts, is in part controlled by the “mechanotransducer” protein YAP (Yes-associated protein), which is shuttled into the nucleus in response to cyto-mechanical forces. Here, using YAP nuclear shuttling as a measurement outcome, we tested the effect of 72 h of clinostat-induced simulated microgravity (SMG) and daily LIV application (LIVDT) on the YAP nuclear entry driven by either acute LIV (LIVAT) or Lysophosphohaditic acid (LPA), applied after the 72 h period. We hypothesized that SMG-induced impairment of acute YAP nuclear entry would be alleviated by the daily application of LIVDT. Results showed that while both acute LIVAT and LPA treatments increased nuclear YAP entry by 50 and 87% over the basal levels in SMG-treated MSCs, nuclear YAP levels of all SMG groups were significantly lower than non-SMG controls. LIVDT, applied in parallel to SMG, restored the SMG-driven decrease in basal nuclear YAP to control levels as well as increased the LPA-induced but not LIVAT-induced YAP nuclear entry over SMG only, counterparts. These cell-level observations suggest that daily LIV treatments are a feasible countermeasure for restoring basal nuclear YAP levels and increasing the YAP nuclear shuttling in MSCs under SMG.

Lamin A/C functions independently from mechanical signaling during adipogenesis

Mesenchymal stem cells (MSC) maintain the musculoskeletal system by differentiating into multiple cell types including osteocytes and adipocytes. Mechanical signals, including strain and low intensity vibration (LIV), are important regulators of MSC differentiation. Lamin A/C is a vital protein for nuclear architecture that supports chromatin organization, as well as mechanical integrity and mechano-sensitivity of the nucleus in MSCs. Here, we investigated whether Lamin A/C and mechano-responsiveness are functionally coupled during adipogenesis. Lamin depletion in MSCs using siRNA increased nuclear area, height and volume and decreased circularity and stiffness, while phosphorylation of focal adhesions and dynamic substrate strain in response to LIV remained intact. Lamin A/C depletion decelerates adipogenesis as reflected by delayed appearance of key biomarkers (e.g., adiponectin/ADIPOQ). Based on RNA-seq data, reduced Lamin A/C levels decrease the activation of the adipocyte transcriptome that is normally observed in response to adipogenic cues mediating differentiation of MSCs. Mechanical stimulation via daily LIV application reduced the expression levels of ADIPOQ in both control and Lamin A/C depleted cells. Yet, treatment with LIV did not induce major transcriptome changes in either control or Lamin A/C depleted MSCs, suggesting that the biological effects of LIV on adipogenesis may not occur at the transcriptional level. We conclude that while Lamin A/C activation is essential for normal adipogenesis, it is dispensible for activation of focal adhesions by dynamic vibration induced mechanical signals.

Low Intensity Vibration Restores Nuclear YAP Levels and Acute YAP Nuclear Shuttling in Mesenchymal Stem Cells Subjected to Simulated Microgravity

Reducing the bone deterioration that astronauts experience in microgravity requires countermeasures that can improve the effectiveness of rigorous and time-expensive exercise regimens under microgravity. The ability of low intensity vibrations (LIV) to activate force-responsive signaling pathways in cells suggests LIV as a potential countermeasure to improve cell responsiveness to subsequent mechanical challenge. Mechanoresponse of mesenchymal stem cells (MSC) which maintain bone-making osteoblasts is in part controlled by the “mechanotransducer” protein YAP (Yes-associated protein) which is shuttled into the nucleus in response cyto-mechanical forces. Here, using YAP nuclear shuttling as a measure of MSC mechanoresponse, we tested the effect of 72 hours of simulated microgravity (SMG) and daily LIV application (LIVDT) on the YAP nuclear entry driven by either acute LIV (LIVAT) or Lysophosphohaditic acid (LPA), applied at the end of the 72h period. We hypothesized that SMG-induced impairment of acute YAP nuclear entry will be alleviated by daily application of LIVDT. Results showed that while both acute LIVAT and LPA treatments increased nuclear YAP entry by 50% and 87% over the basal levels in SMG-treated MSCs, nuclear YAP levels of all SMG groups were significantly lower than non-SMG controls. Daily dosing of LIVDT, applied in parallel to SMG, restored the SMG-driven decrease in basal nuclear YAP to control levels as well as increased the LPA-induced but not LIVAT-induced YAP nuclear entry over the non-LIVDT treated, SMG only, counterparts. These cell level observations suggest that utilizing daily LIV treatments is a feasible countermeasure for increasing the YAP-mediated anabolic responsiveness of MSCs to subsequent mechanical challenge under SMG.

Isolated Nuclei Stiffen in Response to Low Intensity Vibration

ABSTRACTThe nucleus, central to all cellular activity, relies on both direct mechanical input and its molecular transducers to sense and respond to external stimuli. While it has been shown that isolated nuclei can adapt to applied force ex vivo, the mechanisms governing nuclear mechanoadaptation in response to physiologic forces in vivo remain unclear. To investigate nuclear mechanoadaptation in cells, we developed an atomic force microscopy (AFM) based procedure to probe live nuclei isolated from mesenchymal stem cells (MSCs) following the application of low intensity vibration (LIV) to determine whether nuclear stiffness increases as a result of LIV. Results indicated that isolated nuclei were, on average, 30% softer than nuclei tested within intact MSCs prior to LIV. When the nucleus was isolated following LIV (0.7g, 90Hz, 20min) applied four times (4x) separated by 1h intervals, stiffness of isolated nuclei increased 75% compared to non-LIV controls. LIV-induced nuclear stiffening required functional Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, but was not accompanied by increased levels of the nuclear envelope proteins LaminA/C or Sun-2. While depleting LaminA/C or Sun-1&2 resulted in either a 47% or 39% increased heterochromatin to nuclear area ratio in isolated nuclei, the heterochromatin to nuclear area ratio was decreased by 25% in LIV-treated nuclei compared to controls, indicating LIV-induced changes in the chromatin structure. Overall, our findings indicate that increased apparent cell stiffness in response to exogenous mechanical challenge of MSCs in the form of LIV is in part retained by increased nuclear stiffness and changes in chromatin structure.